Background and aims: Peripartum transmission of hepatitis B virus (HBV) from an infected mother to the child can be prevented in most but not all cases by immediate vaccination of the newborn. The aim of this study was to compare the efficacy of two licensed hepatitis B vaccines, Engerix-B versus Sci-B-Vac, in preventing peripartum HBV transmission. Methods: A prospective multicenter randomized controlled study in 4 delivery centers was performed from 2009 to 2014. HBsAg positive pregnant women and their newborns were recruited at the delivery rooms. All newborns received Hepatitis B Immune Globulin within 10 h after birth, as well as active HBV vaccination at 0, 1 and 6 months of age. Maternal assessment at delivery included transaminases, blood count, international normalized ratio and viral status. Infants were tested for HBsAg, anti-HBc and anti-HBs at 12 months of age. Results: In the intention to treat (ITT), 171 infant and mother pairs fulfilled the study enrollment criteria and completed follow up, 82 received Engerix-B and 89 Sci-B-Vac. Maternal parameters and viral status were similar in both groups. At 12 months of age, the Sci-B-Vac group had lower HBsAg carriage rates  than the Engerix-B group  with borderline significance , and borderline significance lower vaccine failure rates with anti-HBs < 10 mIU/mL in the Sci-B-Vac  than in the Engerix-B . Higher seroprotection rates were found in the Sci-B-Vac group with all anti-HBs titer stratifications of >10 mIU/mL (p = 0.05), >100 mIU/mL (p = 0.05) and >1000 mIU/mL (p = 0.01). Active/passive vaccination was effective in 10/13 cases with maternal HBV DNA levels > 7 log10 IU/mL up to 9.5 log10 IU/mL, but failed in 3 cases for unknown reasons. Conclusion: Sci-B-Vac was superior to Engerix-B in preventing peripartum HBV transmission in neonates from HBsAg+ mothers and induces significantly higher anti-HBs levels. NIH registration number: NCT 01133184. The World Health Organization’s (WHO) 2017 hepatitis report indicates that 257 million people are chronically infected worldwide with the hepatitis B virus (HBV) . ImmigraOur study’s goal was to compare the efficacy of these two active hepatitis B vaccines in preventing MTCT in neonates born to HBV carrier mothers.This study was a prospective, randomized, multicenter, open label, parallel group study. Paired participants (HBsAg+ mothers and newborns) were recruited at the delivery rooms of four Israeli hospitals between 2009 and 2014; Dajani Hospital in Jerusalem, Holy Family Hospital in Nazareth, Nazareth Hospital and French Hospital in Nazareth participated in the study. Confirmed HBsAg positive pregnant women (tested by community laboratories) and their newborns were recruited at delivery rooms.The mothers and the newborns were randomly assigned (1:1) into one of two treatment groups using computer based random allocation sequence. The allocation was concealed until the interventions were assigned. The participants were enrolled by the treating physician at each center, while a study coordinator at each center which was blinded to the participants enrolled, randomly assigned participants to either group. After randomizing and assigning treatment, both treating physicians and the participants were unblinded to the type of vaccine administered. All mothers had HBV DNA levels tested at the delivery room. All mothers who were followed by an outpatient hepatologist and who had >7 log10 IU/mL were offered treatment with anti-viral therapy starting at mid-pregnancy around week 20 until 6 months after delivery.Group A newborns received three doses intramuscular (IM) of Engerix-B (10 µg/0.5 mL) within 12 h after delivery, and at ages one and 6 months; group B newborns received three IM doses of Sci-B-Vac (5 µg/0.5 mL) at the same intervals. Cases that by a mistake received different vaccines were excluded, see Serum samples were analyzed for a 500-nucleotide long sequence of the virus polymerase and surface protein nucleotide sequence by polymerase chain reaction (PCR) as described . This geThe study protocol conformed to the ethical guidelines of the 1975 Declaration of Helsinki and was approved by the local Ethics Committee of each institute . Informed consent was waived by the local ethical committee given that the vaccines that were administered at each center was a standard of care practice. All authors had access to the study data and had reviewed and approved the final manuscript.Inclusion criteria included pregnant women who had a chronic HBV infection with HBsAg positivity documented by community laboratories during the first trimester of pregnancy and confirmed in the delivery room , and adhered to the trial protocol, were eligible to participate in the study with newborns. Exclusion criteria included women with other hepatic diseases including, alcoholic liver disease, drug-induced liver injury, autoimmune hepatitis, non-HBV viral hepatitis, cholestatic liver diseases and metabolic/genetic liver disease. Furthermore, we excluded mothers with any other immunity-related disease and those treated with immune suppression. The metabolic background was similar in both groups, including hypertension, diabetes and hyperlipidemia. The delivery room attendance was the first study event for recruitment, screening and randomization timepoints. The previous HBV follow up during pregnancy was variable in the community and not within the study protocol.The primary endpoint was the rate of HBV infections in the infants after 12 months following active vaccination as per the described regimen in the intention to treat analysis (ITT). The rate of HBV infections was defined as the proportion of infants who had detectable serum HBsAg at 12 months of age using the chemiluminescent microparticle immunoassay with Architect Reagent Kits, Abbott.To assess the rates of positive anti-HBc and the vaccine seroprotection rate defined as anti-HBs titer > 10 IU/mL at 12 months of age in the ITT analysis. Moreover, we also aimed to assess possible factors related to the outcome.p-values of 0.05 or less were considered statistically significant. All p-values and confidence intervals were based on two-tailed tests. Wilcoxon rank-sum was used to compare the median values. Regarding sample size calculation, taking into account that almost 87% seroprotection rate of Engerix-B vaccine from real-life data has been demonstrated, we calculated that 87 subjects in each group  are required to have a 80% chance of detecting, as significant at the 5% level, an increase in the primary outcome measure from 87% response rate in the Engerix-B group to 98% in the Sci-B-Vac group.All enrolled newborns were included in the intention to treat analysis of the rates of mother-to-child transmission, as well pre protocol analysis (PPA) was reported. Quantitative variables are expressed as median and range, and categorical variables are expressed in percentages. Categorical variables were compared by applying the Fisher exact test. In the ITT analysis, altogether 183 HBsAg positive pregnant women were identified by community laboratories within the first trimester of pregnancy and were tested again for HBsAg at the time of delivery at the Hadassah Medical Organization laboratory, Jerusalem. Four mothers (2.2%) were excluded, because they had lost HBsAg at the time of delivery. An additional 16 women/newborns were excluded from the study, as 8 babies received two different active vaccines by mistake of whom we have their data regarding HBV status at one year and 8 were lost for follow up early in the study and from whom we lack data. Therefore, in the ITT, a total of 171 women were included in the analysis whereas only 163 mother-infant pairs were included in the per protocol analysis (PPA) because to 8 infants two different vaccines were administered. In the ITT, Group A included 82 newborns who were allocated to the Engerix-B vaccine and all of these infants received the Engerix-B. Group B included 89 newborns, who were allocated to the Sci-B-Vac, of whom 81 received the Sci-B-Vac and 8 received the Engerix-B . There wp = 0.15). The levels of hemoglobin, platelet count, leukocyte count and liver enzymes were similar between the two groups in the ITT and PPA analysis (p = NS), while the INR level were slightly lower in group B  age on delivery in group A was 27.03 ± 9.53 years compared to 28 ± 8.02 years in group B (p = 0.58) in groups A and B, respectively. The median level of HBV viral loads was also similar in the two groups (p = 0.19). In the PPA, the results were almost similar to the ITT analysis  vs. 11/89 (12.4%) of mothers in group A and group B, respectively in the ITT analysis, and 12/82 (14.6%) vs. 10/81 (12.3%) in groups A and B, respectively, in the PPA. On the other hand, 84.1% in group A in the ITT and PPA and 83.1% and 82.7% in the ITT and PPA respectively, had HBV DNA < 5 log10 IU/mL. Of note, 13 women had >7 log10 IU/mL at delivery. They did not receive anti-viral treatment as they had not been monitored by a hepatologist or refused therapy. Sixteen women were treated with nucleoside analogues (NUCs) due to high viremia >7 HBV DNA log10 at weeks 20 of pregnancy. Anti-viral treatment was discontinued 6 months after delivery. Pre-delivery treatment with lamivudine and telbivudine was administered at equal frequencies in groups A and B  and 4 patients were administered tenofovir in the Sci-B-Vac group.In the ITT analysis, the HBeAg positivity rate indicating high HBV infectivity was slightly higher in group B  vs. , whereas there was no statistical difference in the anti-HBe positivity rates . At one year post-delivery, a lower vaccine failure rate was observed in infants in group B (Sci-B-Vac group). More specifically, the proportion of infants who had non-protective anti-HBs titers <10 mIU/mL within one year of delivery was marginally lower among group B compared to group A   .p = 0.05 and OR 4.3, 95% CI 0.9–21.1, p = 0.09), respectively  and 33.3% and 14.8% in the PPA  for groups B and A, respectively. Overall, six newborns were HBsAg-positive at 12 months of age. In the Engerix-B group, one HBsAg positive infant had positive anti-HBs with a titer of 249.8 mIU/mL, while the rest of 5 HBV infected infants had negative anti-HBs. Notably, the use of Sci-B-Vac was associated with a lower incidence of HBsAg positivity as compared to Engerix-B (1.1% and 1.2 vs. 6.1%) in the ITT and PPA, respectively,  , compared to group A (90.1%), respectively,  . Further = 0.05) .10 IU/mL HBV DNA, one mother has 5.5 log10 and two mothers had <1.28 log10. Taken together, active/passive vaccination was effective in 10/13 cases with maternal HBV DNA levels > 7 log10 IU/mL up to 9.5 log10 IU/mL, but failed in 3 cases for unknown reasons. Notably, two cases in group A . Likewise, similar mean maternal HBV DNA levels (p = 0.09) were measured in group A versus B with anti-HBc negative infants  (p = 0.02 and 0.006) higher in the anti-HBc positive infants of groups A & B as compared to the anti-HBc negative infants (The active vaccine type had no significant impact on the rate of anti-HBc positivity at the one year follow up . In the ctively) . However infants .Mother to child HBV transmission (MTCT) is a significant route of HBV transmission, accounting for 40–50% of HBV infections in chronic carriers, particularly in highly endemic areas ,15. MTCTOur study demonstrated that the use of Sci-B-Vac might be more effective in preventing perinatal transmission of HBV mainly of genotype D even from mothers with high viral load, as compared to Engerix-B, as it was associated with significantly higher anti-HBs titers at one-year as compared to the Engerix-B coupled with a trend of significance for a lower HBsAg positivity rate.These results are in line with previous studies. Use of Sci-B-Vac was associated with induction of more rapid and stronger anti-HBs responses in healthy children and newborns .7 IU/mL correlated with a 32% HBV transmission rate compared to 0% transmission rate at HBV DNA viral loads of less than 1.1 × 107 IU/mL [10 IU/m that were treated with NUC’s at mid-pregnancy; none of their babies had detectable HBsAg after one year suggesting the efficacy of HBV screening and antiviral treatment during pregnancy.Several studies have implicated high maternal viremia as the most important factor associated with failure of neonatal vaccination. HBV DNA viral load of more than 1.1 × 1007 IU/mL ,26. Furt07 IU/mL ,28,29. S07 IU/mL . In our 10 > 7 at delivery. In one MTCT case the mother was HBeAg positive with moderately high viremia, log10 = 5.5 which is a level which may lead to MTCT. However, in two cases the HBV DNA assay was negative or very low with log10 ≤ 1.28 with negative HBeAg. This could suggest that a false negative result of the HBV DNA assay in these two cases cannot be excluded or the infant was infected soon after birth from another source because MTCT with undetectable virus loads are virtually unknown [On the other hand, MTCT in this study occurred only in 3 cases  out of the untreated 13 women who had log unknown . Due to The significance of the anti-HBc found in approximately 30% of the infants one year after delivery is not clear. It is probably a remnant of the anti-HBc passively transferred from mother to the baby before delivery. The fact that mothers of anti-HBc positive infants had a slightly higher HBV DNA level could be understood as hint that HBV transmission occurred in some of the infants. But all mothers of HBsAg negative infants had HBV DNA levels < 100.000 IU/mL and were HBeAg negative. Thus, it is more probable that the slightly higher maternal HBV DNA level was associated with a higher maternal anti-HBc level which led to longer detectability of anti-HBc in the infant.Engerix-B contains 10 µg and Sci-B-Vac 5 µg of total HBsAg; both vaccines contain the same adjuvant, use an identical vaccination regimen and both recombinant vaccines originate from HBV genotype A2. The genomic analysis in this study did not find any vaccine escape mutations which could explain the observed failures of Engerix-B. The maternal HBV genotyping demonstrated mostly subgenotype D1 in our cohort. Differences in the vaccine genotype and the infecting virus may reduce protection as observed in newly infected blood donor candidates of the US Red Cross  and may The limitations of our study include that all pregnant women were recruited into the study at the delivery room, as this led to imbalance of treating women with high viremia during pregnancy with antivirals (6/82 in the Engerix-B vs 10/89 in the Sci-B-Vac groups), the second limitation is that the difference was marginally significant, and the last one is that we analyzed only small number of samples for genetic testing due to the fact that we could not obtain more peripheral blood samples from the other recruited mothers.In conclusion, Sci-B-Vac might be more effective than Engerix-B in preventing HBV infection of genotype D in early childhood and may contribute to achieve the WHO goal for global elimination of HBV infection as a public health hazard. Further studies are needed to validate our findings.


There is a lack of population level data on risk factors, incidence and impact of SARS-CoV-2 infection in pregnant women and their babies. The primary aim of this study was to describe the incidence, characteristics and outcomes of hospitalized pregnant women with symptomatic and asymptomatic SARS-CoV-2 in the UK compared to pregnant women without SARS-CoV-2.We conducted a national, prospective cohort study of all hospitalized pregnant women with confirmed SARS-CoV-2 from 01/03/2020 to 31/08/2020 using the UK Obstetric Surveillance System. Incidence rates were estimated using national maternity data. Overall, 1148 hospitalized women had confirmed SARS-CoV-2 in pregnancy, 63% of which were symptomatic. The estimated incidence of hospitalization with symptomatic SARS-CoV-2 was 2.0 per 1000 maternities (95% CI 1.9–2.2) and for asymptomatic SARS-CoV-2 was 1.2 per 1000 maternities (95% CI 1.1–1.4). Compared to pregnant women without SARS-CoV-2, women hospitalized with symptomatic SARS-CoV-2 were more likely to be overweight or obese  and aOR 2.07 (1.53–2.29)), to be of Black, Asian or Other minority ethnic group , aOR 4.36, (3.19–5.95) and aOR 12.95, (4.93–34.01)), and to have a relevant medical comorbidity (aOR 1.83 (1.32–2.54)). Hospitalized pregnant women with symptomatic SARS-CoV-2 were more likely to be admitted to intensive care ) but the absolute risk of poor outcomes was low. Cesarean births and neonatal unit admission were increased regardless of symptom status  and aOR 3.08, (1.99–4.77); asymptomatic aOR 2.02, (1.52–2.70) and aOR 1.84, (1.12–3.03)). The risks of stillbirth or neonatal death were not significantly increased, regardless of symptom status.We have identified factors that increase the risk of symptomatic and asymptomatic SARS-CoV-2 in pregnancy. Clinicians can be reassured that the majority of women do not experience severe complications of SARS-CoV-2 in pregnancy. In March 2020 the World Health Organization declared a global pandemic of novel coronavirus infection (SARS-CoV-2) . Evidencth May 2020 . Wo. Wo11]. st November 2017 and 30th October 2018 were identified ) who could be over or underrepresented in the analysis by ethnic groups. Alternatively, rates of smoking in pregnancy vary across the UK (from 1.6% in Wokingham to 27.8% in Blackpool[23]) so it is plausible this could represent measurement bias, if places with lower smoking rates had higher rates of admission with SARS-CoV-2. Plausible biological explanations also exist, for example, nicotine may exert an anti-inflammatory effect and inhibit the production of pro-inflammatory cytokines, alternatively increased nitric oxide may inhibit the replication of SARS-CoV-2 at cell entry[24].” In the last paragraph of the introduction, the sentence containing “….risk of infection with SARS-CoV-2 or resulting morbidity  to explore if any were…” suggests that the authors were investigating diabetes as a resulting morbidity.This has been reworded as follows:Line 171-2: “A sensitivity analysis was undertaken to explore if any pre-existing medical problems (e.g. diabetes), that might have increased the risk of infection with SARS-CoV-2 or resulting morbidity, were independently associated with the outcome.” In both the introduction and the discussion sections, the authors describe their study results as unbiased. There may indeed be a bias in the reported data if getting to hospital and being tested is associated with women’s characteristics and underlying risk of exposure to the virus.This measurement bias has already been explored in the discussion in lines 385-393. The wording in the discussion has been altered to remove the word ‘unbiased’. The use of the word ‘unbiased’ in the introduction is in description of the available evidence and therefore has not been changed: “However, the majority of studies to date are case reports, case series and institutional or registry non-population-based cohort studies and there is a lack of population-level data to inform accurate incidence rates and unbiased descriptions of characteristics and outcomes.” With respect to this potential bias, it would be useful for the authors discuss their findings for pre-existing conditions. Differences in results between symptomatic and asymptomatic women are interesting.The sentence that describes these differences in the results has been expanded:Line 212-213: “In the sensitivity analysis, there was some evidence that asthma and hypertension specifically increased the risk of admission with symptomatic SARS-CoV-2, although the numbers in some groups were small PONE-D-21-00575R1Dear Prof. Knight,We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at onepress@plos.org.If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact Kind regards,Diane FarrarAcademic EditorPLOS ONEAdditional Editor Comments:As you will be aware there is a move away from using p values, as many results are not appropriately dichotomized in this way, however readers may use p values as a guide and some may have limited knowledge of the meaning of some estimates, therefore I would be happy for p values to remain in the tables. Also please leave the tables in their present format, I agree that amalgamating them would result in overly large tables. Would you consider changing 'pregnant mothers' to 'pregnant women', abstract and intro and throughout the manuscript when referring to antenatal women, both terms are used and one should be chosen for consistency to describe women before they have given birth, post birth, women can be referred to as mothers of course. 28 Apr 2021PONE-D-21-00575R1 The incidence, characteristics and outcomes of pregnant women hospitalized with symptomatic and asymptomatic SARS-CoV-2 infection in the UK from March to September 2020: a national cohort study using the UK Obstetric Surveillance System (UKOSS) Dear Dr. Knight:I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. onepress@plos.org.If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. 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The presence of the human leukocyte antigen HLA-B*57:01 is associated with the development of a hypersensitivity reaction to abacavir (ABC). Limited data exist on HLA-B*57:01 prevalence in individuals with HIV-1 in Africa. This study aimed to estimate HLA-B*57:01 prevalence in individuals with HIV-1 in West and Central Africa.A cross-sectional study was conducted in four countries in West and central Africa  from January 2016 to February 2020 to determine the status of HLA-B*57:01 in adults with HIV-1. The presence of HLA-B*57:01 was determined by using Single Specific Primer-Polymerase Chain Reaction (SSP-PCR) in blood samples. Prevalence rates were stratified based on country.A total of 4016 (69.8% women) individuals with HIV were enrolled. Their median age was 45, and the interquartile range was 38–52. We included 500 (12.4%) patients in Burkina-Faso, 1453 (36.2%) in Côte d’Ivoire, 951 (23.7%) in Gabon, and 1112 (27.7%) in Togo. The overall HLA-B*57:01 prevalence was 0.1% [95% CI: 0.0–0.2%]. The prevalence of HLA-B*57:01 was similar according to the four countries. Only one case was reported in each country except Togo, with no cases.HLA-B*57:01 prevalence is low in individuals with HIV in West and central Africa, and there is no difference among countries. This study does not confirm the utility of HLA-B*57:01 allele testing for abacavir use in this region. P = 0.01) ), and more than two-thirds (69.8%) were female. Most participants were 40–49 years old (38.0%), and 46.9% had at least a secondary school level of education. The majority of patients were on ART (More than two-thirds 62.4%) had been diagnosed with HIV for more than 5 years (at the time of the survey). A higher proportion of them (90.9%) had HIV-1 status, and approximately one-quarter (26.3%) were at WHO stage I. Other demographic, clinical, and laboratory characteristics of all participants by country are presented in Table .4% had bp = 0.238), sex (p = 0.078), or age (p = 0.276) . There was no significant difference in the prevalence of the HLA-B057 allele according to country . One case was reported in Côte d’Ivoire, Gabon and Burkina Faso, and no case was reported in Togo Table .n = 3) and Central (n = 1) African regions of the prevalence of the HLA-B*57:01 allele among individuals with HIV. According to the latest epidemiological data, West and Central Africa is home to 4.9 million people living with HIV. HIV prevalence among adults is 1.4%, which is relatively low compared to East and Southern Africa. In this region, the number of people accessing treatment rose significantly from 860,000 in 2010 to 2.9 million in 2019. HLA-B*57:01 is a generic marker of clinical importance used in several countries, specifically in developed countries, to decrease abacavir-related hypersensitivity reactions. Guidelines in North America and Europe recommend routine screening for HLA-B*57:01 prior to initiation of abacavir therapy [To our knowledge, this study is the first large multicountry study conducted in four countries in the West  were HLA-B57 positive using a nonspecific low-resolution HLA-B*57:01 primer mix. On further analysis, none of the 132 samples (0%) had the HLA-B*57:01 allele . AnotherOur study had a few limitations. We did not conduct an analysis based on race in our survey. Other races are indeed rare in individuals with HIV in West and central Africa, and this variable is not collected routinely in databases. Further studies should explore including race , as in our sample, one person in Gabon with the mestizo race was screened positive. Some studies have reported that the heterogeneity of HLA-B*57:01 prevalence is mostly dependent upon race and ethnicity heritage , 33. AnoSince abacavir is included in the first-line regimen, the question of hypersensitivity to abacavir should be important to document. However, to our knowledge, there are no guidelines for screening patients before ART initiation in Africa. Before abacavir should be wisely used in Africa, it is necessary to explore the prevalence of HLA-B*57:01. The application of routine HLA-B*57:01 testing to other racial populations deserves further investigation. In these four countries, antiretroviral therapy is available and free of charge for patients. Currently, the first-line regimen includes 2 NRTIs and dolutegravir in accordance with the WHO guidelines or 2 NRTIs and efavirenz. An optimized NRTI backbone should be used, such as zidovudine (AZT), tenofovir or abacavir (ABC), and vice versa.In addition, HLA-B*57:01 screening was not performed routinely because of the lack of equipment and reference laboratories specializing in immunologic tests in the four countries. This is the main reason all-immunological analyses were performed in Belgium, which is specialized in this analysis to avoid heterogenicity between the tests.Recently, the value of HLA-B*57:01 screening prior to prescribing abacavir is a concern in areas where its prevalence is low , 35. In HLA-B*57:01 is rare in people living with HIV, in the four countries participating to this survey, and there are no significant differences among countries. HLA-B*57:01 screening may not be a cost-effective strategy to reduce the risk associated with abacavir hypersensitivity in these countries. Indeed, the prevalence of HLA-B*57:01 allele positivity was low in the study population. These results cannot be generalized to the West and Central African region as prevalence may be much higher in other countries and among other ethnicities in the same regions of Africa.However, despite the low prevalence of the HLA-B*57:01 allele, rigorous monitoring should be maintained in any patient on ABC since the possibility of occurrence of HSR to this drug should not be definitively ruled out.


This study is aimed at teasing out the correlation of plasma D-dimer (D-D) levels to age, metastasis, TNM stage (tumor-node-metastasis classification), and treatment in non-small-cell lung cancer (NSCLC) patients of different ages, to facilitate early diagnosis of hypercoagulable state, choose appropriate treatment, and use appropriate anticoagulants. Hence, thrombosis and complications caused by excessive anticoagulants can be prevented; thrombus or disseminated intravascular coagulation (DIC) and other complications in elderly patients with NSCLC can be reduced or avoided. By monitoring the level of plasma D-D in patients with NSCLC, recurrence and metastasis can be predicted in the early stage and the TNM stage can be evaluated.  A total of 670 patients with NSCLC were selected in Shanxi Bethune Hospital from March 2014 to October 2020 as the experimental group, and 950 healthy people were selected from the physical examination center of the same hospital as the control group. The data of patients with NSCLC diagnosed for the first time without any treatment were collected and grouped based on metastasis, TNM stage, treatment, and pathological type, and the correlation with plasma D-D level was analyzed. Plasma D-D levels were measured by immunoturbidimetry on an ACL TOP 700 Automatic Coagulation Analyzer. The patients were further divided into two groups according to different treatment methods, and the differences in plasma D-D levels between patients receiving chemotherapy and those receiving targeted therapy in different treatment cycles were analyzed. The correlation between D-D levels and age in healthy controls was analyzed. The difference in D-D levels between NSCLC patients and healthy controls of the same age was analyzed. p > 0.05). The plasma D-D level of NSCLC patients was significantly higher than that of the healthy controls (p < 0.05). No significant difference in plasma D-D level was found between NSCLC patients of different genders, and the finding was similar between healthy controls of different genders (p > 0.05). Significant difference in the D-D level was found between the groups of 30-59 years and 60-69 years (p < 0.05), between groups of 60-69 years and 70-79 years (p < 0.05), and between 70-79 years and ≥80 years (p < 0.05). The plasma D-D level of patients ≤ 79 years old increased with age, but it decreased in those over 80 years old. According to Pearson correlation analysis, there was a positive correlation between the D-D level and the age of NSCLC patients under 79 years old (p < 0.05). The differences in D-D levels between the four age groups were statistically significant (p < 0.05), showing an upward trend of the D-D level in healthy controls with the increase of age. There were statistically significant differences in D-D levels between NSCLC patients and healthy controls of the matching age group (p < 0.05), suggesting that NSCLC patients had significantly higher D-D levels than healthy people of the same age group. The differences in D-D levels between NSCLC patients without metastasis, NSCLC patients with metastasis, and healthy people were statistically significant (p < 0.05). The patients with metastasis had the highest D-D level, and healthy people had the lowest D-D level. The difference in plasma D-D levels between patients of different TNM stages was statistically significant (p < 0.05). Patients with an advanced TNM stage tended to have higher D-D levels. The TNM stage and D-D level of NSCLC patients changed significantly before and after treatment. An earlier stage was related to a more obvious change in D-D levels after treatment with a statistically significant difference (p < 0.05). A more advanced stage was associated with a smaller change in the D-D level after treatment, with no statistically significant difference (p > 0.05). The plasma D-D levels before and after four cycles of chemotherapy or targeted therapy were higher than those of the healthy control group, and the differences were statistically significant (p < 0.05). The D-D level of patients after chemotherapy was significantly lower than that before chemotherapy (p < 0.05), but there was no significant difference before and after targeted therapy (p > 0.05). The D-D level after the first cycle of chemotherapy was higher than that before chemotherapy. The level of D-D after the third and fourth cycles was significantly lower than that before chemotherapy (p < 0.05). No significant difference was found between the D-D level before treatment and that after four cycles of chemotherapy (p > 0.05).  All data of both the experimental group and the control group were normally distributed. The average age of the experimental group was 61.31 ± 6.23 (range: 36-92) years. The average age of the control group was 61.14 ± 11.12 (range: 35-85) years. There was no significant difference in gender between the experimental group and the control group ( It is suggested that coagulation test indexes should be included to evaluate the treatment regimen for NSCLC patients. Most patients with NSCLC are in a hypercoagulable state, which is related to age, tumor invasion and metastasis, recurrence, and treatment. Regular monitoring of plasma D-D levels can facilitate early diagnosis of a hypercoagulable state and timely and appropriate use of anticoagulants, to avoid or reduce complications such as venous thromboembolism in NSCLC patients and to prevent the risk of bleeding caused by excessive anticoagulants. Clinicians can choose the treatment with less harm and maximum benefit for NSCLC patients based on the plasma D-D level. When in a hypercoagulable state, the body's blood viscosity increases, making it more conducive to the growth and infiltration of tumor cells. Our study shows that the recurrence and metastasis of NSCLC are related to coagulation indexes, which provides a theoretical basis for the early diagnosis and treatment of recurrent and metastatic NSCLC. Lung cancer is the most common and deadly malignant tumor worldwide. At the time of diagnosis, 80% of lung cancer patients are in the advanced stage, and the 5-year survival rate is less than 15%, posing a serious threat to human health . Non-smaThe hypercoagulable state, which is common in patients with malignant tumors, can result in thrombosis, such as pulmonary embolism, and hence is a leading factor of death –7. In re670 NSCLC cases and 950 healthy volunteers were selected for the investigation. All of them were in the Shanxi Bethune Hospital from March 2014 to October 2020. The NSCLC patients were diagnosed according to the World Health Organization criteria (2015 edition). And their TNM staging is based on the International Association for the Study of Lung Cancer (IASLC) and the Union for International Cancer Control (UICC) (issued in January 2017) . HealthyNSCLC patients of inclusive and exclusive criteria: inclusive: age group: 35 to 95 years old, male or female; firstly diagnosed by pathology or cytology; expected survival life-time ≥ 3 months; without other cancers; volunteer; and no enrollment in other clinical trials. Exclusive: with severe immunodeficiency or autoimmune diseases; with chronic congestive heart failure; with a history of operation, trauma, and burn in six months; and with hormone or anticoagulant therapiesTNM staging(4) NSCLC patient therapy grouping2 (d1) + cisplatin  75 mg/m2 (d1-d5), ivgtt, was given according to the body surface area. Oral folic acid 400 μg/d was given 7 days before the first administration of pemetrexed for at least 5 days until 21 days after the last administration, and vitamin B12 1000 μg was injected intramuscularly 7 days before the beginning of the first cycle of chemotherapy, then once every 3 cycles. Dexamethasone tablets  were taken orally on the day before, the same day, and after administration to prevent skin reaction. The above chemotherapy consists of one cycle every 3 weeks, with a total of four cycles. If the disease progressed or there were serious adverse reactions, the treatment was stopped and the study was withdrawn, but the adverse reactions were recorded. Targeted drug therapy group: the targeted treatment group was treated with erlotinib ). After continuous treatment for 3 weeks, the interval was 1 week for 1 cycle, and the treatment was carried out for 4 cycles. (5) Healthy volunteers of inclusive criteriaChemotherapy group: chemotherapy regimen: on the basis of supporting treatment, pemetrexed  500 mg/m(6) Age grouping Age group: 35 to 95 years old, male or female; Negative tumor markers; Healthy (conclusion) with the normal result of accessory examination; Volunteer; No enrollment in other clinical trials. r = 13 cm), 25°C. NSCLC patient's blood samples were collected 12 hours before and after treatment.Plasma sample: fasting elbow venous blood was collected  and centrifugated at 3500 r/min for 10 min  XL Kit, Liquid Antithrombin, D-D HS500). The normal range of Pt was 9 s-12 s; the normal range of PTA was 80%-160%; the normal range of INR was 0.8-1.4; the normal range of APTT was 28 s-41 s; the normal range of FIB-C was 2.00 g/l-4.40 g/l; the normal range of D-D was 0-200 ng/l.t/F test. The qualitative data (or counting data) were described by percentage, and the comparison between groups was described by percentage χ2 inspection. Pearson correlation was used to analyze the correlation. The difference was statistically significant (p < 0.05).All data were analyzed by SPSS 19.0 statistical software, and the measurement data in accordance with normal distribution were analyzed by means ± standard deviationp > 0.05). A significant difference was found in the number of cases of different ages between the two groups (p < 0.05), which was comparable. Compared with the control group, the levels of D-D (D-dimer), PT (Prothrombin Time), INR , and FIB-C (Fibrinogen-c) in the experimental group were significantly higher (p < 0.05), the level of TT (Thrombin Time) was significantly lower (p < 0.05), and the levels of PT% (Prothrombin Time Activity) and APTT  were significantly lower (p > 0.05) , respectively. There was no significant difference in the number of different genders between the two groups (p < 0.05), and no significant difference was found between the other age groups (p < 0.05). The D-D levels of 30-59, 60-69, and 70-79 age groups showed an overall upward trend with increasing age, but the D-D level of the ≥80 age group was lower than that of the 60-69 age group and the 70-79 age group. Pearson correlation analysis showed that the D-D levels of 30-59, 60-69, and 70-79 age groups were positively correlated to age (p < 0.05), indicating that in patients younger than 80 years, D-D levels presented an upward trend with the increase of age . Pearson correlation analysis showed a positive correlation between D-D level and age in all age groups (p < 0.05). The level of D-D in the control group showed an upward trend with the increase of age in the controls younger than 80 years old . The D-D level in all age groups of the experimental group was significantly higher than that in all age groups of the control group. The D-D level showed an upward trend with the increase of age in subjects younger than 80 years old in both groups. However, for those older than 80 years in the experimental group, the D-D level decreased . Therefore, it was the D-D level in patients with metastasis (p < 0.01). The D-D level of patients without metastasis was significantly higher than that of those with metastasis (p < 0.01) (There were 98 patients without metastasis and 572 patients with metastasis. The D-D level of patients without metastasis was significantly higher than the control group ( < 0.01) .p < 0.001)  .p > 0.05), but a statistically significant difference was found when comparing the D-D level of the control group with that of stages II, III, and IV patients (p < 0.05). There was a significant difference in the D-D level between stage I and stage II patients (p < 0.05) (p < 0.05) (p < 0.05) (p < 0.05), indicating that the D-D level presented an upward trend with the increase of TNM classification  . The D-D < 0.05) . The D-D < 0.05) . Pearsonfication .p < 0.05) (p < 0.05)  was significantly lower than that before treatment ( < 0.05) . However < 0.05) .p < 0.05); the D-D level after treatment was significantly lower than that before treatment (p < 0.05) (p < 0.05) (p < 0.05), indicating that D-D levels presented a downward trend with the increase of chemotherapy cycle (p < 0.05)  . The D-D < 0.05) . Pearsonpy cycle . The dif < 0.05) .p < 0.05) (p > 0.05) (p > 0.05) (p > 0.05) (p < 0.05), indicating that D-D levels presented a downward trend with the increase of targeted therapy cycles  . The D-D > 0.05) . The D-D > 0.05) . The com > 0.05) . Pearsony cycles .The incidence of NSCLC ranks first among all tumors, and it is more commonly found in the elderly . HoweverStudies have shown that the survival rate of malignant tumor patients without venous thromboembolism (VTE) is at least 3 times higher than that of patients with VTE, and the risk of death for tumor patients with VTE can be increased by 2-6 times . The incAge is often used as an important reference index. Studies on the relationship between D-D levels and age in healthy people have shown that healthy elderly people (≥60) have a significantly higher D-D level than healthy young people . The incα) damage the vascular endothelial cells, resulting in the hypercoagulable state of blood [Increasingly clinical evidence has shown obvious abnormalities in the coagulation system and significantly increased D-D levels in patients with malignant tumors, such as digestive system malignant tumor, liver cancer, colorectal cancer, gastric cancer, and breast cancer. The mechanism may be that tumor cells or necrotic surrounding tissues trigger the release of tissue factors, which activate coagulation factors X and XI in the extrinsic coagulation system and fibrinolytic system, leading to the local production of fibrinolytic enzymes, which can directly degrade the extracellular matrix and endow the tumor cells with the invading and metastatic abilities . In thisof blood , 28 and of blood . As a spp < 0.05). There was no significant difference in the level of D-D between stage I and stage II patients (p > 0.05). The D-D level of stage III patients was significantly higher than that of stage I and II patients (p > 0.05). The main reason may be that the tumor infiltration of stage III and IV patients is deeper and more extensive than that of stage I and II patients and is often accompanied by lymph nodes and other distant metastases. Cancer patients often suffer complications, such as hypertension, hyperlipidemia, heart failure, and myocardial infarction, which can accelerate blood coagulation, making the D-D level higher. Therefore, monitoring D-D levels and improving the hypercoagulable state of blood in NSCLC patients of different TNM stages can facilitate the early prevention and treatment so as to increase the survival rate of patients. In addition, a majority of NSCLC patients are elderly, and elderly patients, especially those with advanced cancer, are usually bedridden for a long time, resulting in blood stasis. Therefore, NSCLC patients are more likely to suffer abnormal coagulation function and thrombosis than healthy people [This study found that the D-D level in NSCLC patients was significantly higher than that in the control group, which can be explained as follows. The tissue of NSCLC patients can secrete procoagulants, and their antithrombotic ability decreases after the vascular intima is infiltrated by lung cancer tissue, both resulting in hypercoagulability in patients . Cancer y people .NSCLC is a malignant tumor with high mortality. In clinical practice, thrombolysis, anticoagulation, and other treatments usually start after obvious thrombosis appears in patients, and therefore, the curative effect is poor. Many patients die of serious thromboembolism, disseminated intravascular coagulation, and other adverse events . ChemothThis study found that the plasma D-D level of NSCLC patients was higher than that of healthy people, with significant differences, indicating that NSCLC patients are in a hypercoagulable state, which can cause VTE at any time . This reThis study also found that after chemotherapy, the difference in the D-D level between two consecutive cycles was not significant, but a significant difference was found between the two cycles with a one-cycle interval, and the difference before and after treatment was also significant, indicating that chemotherapy can well control the hypercoagulable state of NSCLC patients. In patients receiving targeted therapy, the D-D level showed an overall downward trend after treatment. Although there was no significant difference in the first four cycles, the difference was increasingly obvious. Our results suggest that targeted therapy has an impact on the D-D level of NSCLC patients, but chemotherapy exerts a better effect on the improvement of hypercoagulable state in patients. In addition, heparin can be administered when necessary based on the D-D level of patients  in orderThe plasma D-D level is conducive to the early detection and treatment of NSCLC. For NSCLC patients, blood coagulation function should be examined regularly to detect the hypercoagulable state of blood and conduct treatment timely. An older age is associated with a higher D-D level, advanced stage, and metastasis in NSCLC patients. The elevated D-D level indicates the existence of a hypercoagulable state and the activation of the secondary fibrinolytic system, which can increase the risk of VTE and disseminated intravascular coagulation. Therefore, monitoring the D-D level can help to judge and predict the disease condition. To treat NSCLC, we can use antithrombotic, low molecular weight heparin, and other preventive anticoagulants to eliminate or reduce the risk of deep venous thrombosis and other complications and to control tumor metastasis and improve the comprehensive curative effect and survival of patients.


Blood samples were tested by enzyme-linked immunosorbent assay D-5501 SARS-Cov-2-IgG-EIA-BEST and D-5502 SARS-Cov-2-IgM-EIA-BEST . The overall seropositivity rate was 28.33–28.53%. SARS-CoV-2 antibodies were detected in 17.23%  of participants in the high-risk and 45.95%  in the symptomatic group. Higher IgG and IgM titers were observed in women compared to men, as well as in participants in the symptomatic group compared to those in the high-risk group. The results indicate that the seroprevalence among residents in several Russian regions is low (28.38%) and inadequate to provide herd immunity. The lower seroprevalence among participants in the high-risk group may be attributed to the enforcement of healthcare protocols and the use of adequate personal protective equipment.The present study aimed to assess antibody seropositivity prevalence among symptomatic individuals and individuals with a high risk of occupational exposure to SARS-CoV-2. Participants from Chelyabinsk (Russian Federation) who were at an increased risk of exposure to SARS-CoV-2 (high-risk group,  The coronavirus disease 2019 (COVID-19) pandemic has spread rapidly to more than 180 countries worldwide, resulting in high levels of morbidity and mortality. As of 9 May 2021, nearly 166 million COVID-19 cases and 3449 million deaths have been reported [The clinical manifestations of COVID-19 can range from an asymptomatic/mild disease to severe disease with acute respiratory tract infections. Data from the meta-analysis suggest that the pooled prevalence of asymptomatic COVID-19 is about 48% and is higher in females than in males . With thWhile CDC does not recommend serology testing to diagnose the current infection , its utiThe seroconversion of specific SARS-CoV-2 IgG/IgM antibodies can start as early as 4 days , with thA considerable amount of literature has been published on the impact of the pandemic on healthcare workers , while tAlthough the COVID-19 outbreak in Russia started later compared to many neighboring European countries, Russia is currently among the six countries with the highest number of confirmed COVID-19 cases, as of 9 May 2021 ,23. LimiThus, the present pilot study aimed to assess and compare antibody seropositivity prevalence rates among symptomatic individuals and individuals in occupations with a high risk of exposure to SARS-CoV-2.Chelyabinsk is the seventh-largest city in Russia with a population of approximately 1.3 million. The city has a humid continental climate (Köppen: Dfb): the average temperature in January is −14 °C/6.6 °F. and 19 °C/66.7 °F in July. The first case in the city was registered on 21 March 2020, and 58,380 cases with 1417 deaths from COVID-19 were reported as of 24 April 2021.In this cross-sectional study, we assessed the prevalence of SARS-CoV-2 infection via serological testing for anti-SARS-CoV-2 antibodies. A total of 1300 persons, who were at an increased risk of exposure to SARS-CoV-2 , were invited to participate in a free employer-sponsored SARS-CoV-2 serology assessment between 28 September 2020 and 30 December 2020.This study excluded patients younger than 18 years with a history of coronary heart disease, pre-excitation syndromes, motor impairments , with pacemakers, drug addicts, and dialysis patients.One thousand and ninety-one persons were enrolled in the study and comprised the “high-risk” group; 209 persons did not agree to participate or did not attend the hospital for a scheduled appointment. Individuals were excluded from this group if they reported symptoms of COVID-19 or other acute respiratory virus infections on the day of the appointment.Six hundred and ninety-two persons who had symptoms, reported having symptoms consistent with COVID-19 or other acute respiratory virus infections , were suspected to have COVID-19 infection in the past, or had contact with COVID-19 infected persons and self-reported for enrolment in the study comprised the “symptomatic/contact group”.The study proposal and protocol were approved by the ethics committee of the Institute of Health “DoctorLab” (1 July 2020). Reporting was in accordance with the Strengthening the Reporting of Observational Studies in Epidemiology guidelines. A representative of the research team approached the individuals by phone to ask if they would be willing to have a research coordinator speak to them about the study. If yes, the coordinator spoke with them, described the study . Individuals were given adequate time to reflect on the information, had any questions answered, and gave free and voluntary consent. Patient consent forms were distributed to the participants at reception areas of the Institute of Health “DoctorLab” (LLC “DoctorLab”).® SST™ Tubes containing spray-coated silica and a polymer gel for serum separation  and centrifuged at 1500× g for 20 min. The obtained blood serum was used for the IgM and IgG detection on the day of venipuncture. Serum samples were tested by enzyme-linked immunosorbent assay (ELISA) D-5501 SARS-Cov-2-IgG-EIA-BEST and D-5502 SARS-Cov-2-IgM-EIA-BEST . All samples were tested in duplicate. The test was performed according to the manufacturer’s instructions [Peripheral blood was collected by venipuncture in BD Vacutainerructions .2SO4), absorbance at 450 nm with a 620-nm reference was measured in an ELISA plate reader. The intensity of the staining is proportional to the concentration of IgG (IgM) to SARS-CoV-2 in the analyzed sample. The total procedure requires 10 μL of plasma and the duration of the assay is about 2 h.The method of determination is based on a two-stage “indirect” version of ELISA. At the first stage of the analysis, the specific antibodies (IgG or IgM) contained in the test samples bind to the recombinant SARS-CoV-2 antigen immobilized on the surface of the plate wells—receptor-binding domain (RBD) of glycoprotein S . At the second stage, the conjugate of monoclonal antibodies to human IgG (IgM) with horseradish peroxidase interacts with antigen–IgG (antigen–IgM) complexes. During incubation (25 min) with a tetramethylbenzidine solution, the solution stains in the wells contained the formed antigen–IgG-conjugate complexes. After stopping the reaction by addition of the stop solution  calculations to display the antibody content were carried out following the manufacturer’s instructions . CP show−) were calculated. The results were accounted for if the following conditions were met—the average OD value in the well with K was not more than 0.2—OD value in the well with K+ was not less than 0.5.For this, the arithmetic mean values of optical density in the wells with a negative control sample  was calculated by the formula:CP was calculated using the formula:The test results were considered positive if CP was equal or more than 1.1, negative—if CP was less than 0.8, doubtful—if the results were between 0.8 and 1.1.Data management and analysis were carried out using software R 3.1.1 12  and Microsoft Excel version 14.0. Since all selected groups of the general sample had an abnormal distribution (Shapiro–Wilk test < 0.05), the statistical criteria chosen for the calculations were nonparametric. To study the correlation between the age of patients and the concentration of immunoglobulins, the Spearman rank test was chosen. The Kruskal–Wallis rank test was used to compare the concentration of immunoglobulins between men and women and between individuals from the “high-risk” and “symptomatic” groups.Samples from 1101 men and 682 women with an average age of 39 years were investigated . Of the Blood samples were tested for the presence of both IgM and IgG with the above-mentioned ELISA kits. The detailed distribution of the seropositivity rates is presented in Among the 1091 persons from the “high-risk” group, positive results for the presence of SARS-CoV-2 antibodies were detected in 17.23% of cases , doubtful results—in 0.73% of cases .Among the 692 persons from the “symptomatic” group, positive results for the presence of SARS-CoV-2 antibodies were detected in 45.95% of cases , doubtful results—in 1.01% of cases.The crude seropositivity rate in the total sample was 28.38% ; doubtful results were obtained in 0.84% . With thThe measurement errors of tests can result in biased prevalence estimates, and thus, the adjusted seropositivity rates were calculated  dependinAccording to the conducted calculations, the adjusted seropositivity in the total sample was 28.33–28.53% .Adjusted seropositivity was significantly higher in the “symptomatic” group—45.91–46.24% compared to the values (17.17–17.29%) of the “high-risk” group. Seropositivity for two immunoglobulins (the presence of IgM and IgG to SARS-CoV-2) was 10.29–10.37% in the “high-risk” group and 32.03–32.26% in the “symptomatic” group. Seropositivity of individual IgM-positive and IgG-positive patients from the “high-risk” group was 2.21–2.57% and 4.25–5.94%, respectively. For the “symptomatic” group, a similar calculation of seropositivity revealed the higher values: 9.17–12.81% for IgG-positive and 4.42–5.14% for IgM.Adjusted seroprevalence of antibodies to SARS-CoV-2 in women was 45.91–46.24%  and in men 17.17–17.29% .The average content of IgM antibodies in the total sample was 1.59, and the average content of IgG was 3.17.p-value = 0.0004) and between age and IgG levels . A statistically significant positive relationship was also found between the IgG and IgM levels .According to the Spearmen test, the statistically significant positive relationship was found between age and IgM levels  and subjects from the symptomatic group compared with those from the high-risk group .According to the Mann–Whitney test, the higher IgG and IgM levels were observed in women compared with men  and levels of SARS-CoV-2 antibodies in participants from the symptomatic group were significantly higher than those in the high-risk group. These results match other studies  and are Existing evidence suggests that seroprevalence worldwide is equivalent between sexes ,17. MoreThus, consistent with the literature, this research found that antibody levels were higher in women compared to men, as well as in older versus younger participants ,37,39,40However, the contemporary clinical data are rather controversial, and there is no general agreement about the impact of sex on antibody generation and prognosis in SARS-CoV-2 infection ,50,51,52The obtained results are in accord with recent studies indicating that average IgG/IgM antibody levels were higher in old ages ,53. ThesThe herd immunity threshold for SARS-CoV-2 is approximately 67% . The resThis study had several limitations due to the scale of the COVID-19 pandemic. First, this study was limited to a single city. Therefore, the results may not be generalizable to other regions that are more geographically diverse. Furthermore, due to the regulation rules, persons aged >65 years were not permitted to work during the pandemic; this may have affected the seropositivity rates observed in this study. Second, as random sampling was not used, the estimated seroprevalence was subject to potential sampling bias. Third, samples collected from infected individuals outside the antibody response time window and low diagnostic rates of the used commercial test systems in the initial phase of infection may have yielded false-negative results; therefore, the observed seroprevalence in our study may have underestimated the true prevalence rate of COVID-19. Fourth, we did not evaluate dynamic changes in antibody titers in infected individuals over time, and depending on the sampling time, there might be a higher incidence of seropositive individuals in the symptomatic group. Fifth, the prevalence estimates may change with new information on the accuracy of the test kits that we used. Sixth, since the majority of COVID-19 patients are either asymptomatic or have only a few mild symptoms, the sensitivity of the antibody tests in the general population may be lower; this may affect the reliability of antibody-based epidemiological studies .


The major limitations of curcumin are its low oral bioavailability and difficulty in permeating the blood–brain barrier. Therefore, designing a delivery system of curcumin to overcome its limitations must be employed. KLVFF, a peptide known as an amyloid blocker, was used in this study as a targeting moiety to develop a targeted drug delivery system. A prototype of transnasal KLVFF conjugated microemulsions containing curcumin (KLVFF-Cur-ME) for the nose-to-brain delivery was fabricated. The KLVFF-Cur-ME was developed by a titration method. A conjugation of KLVFF was performed through a carbodiimide reaction, and the conjugation efficiency was confirmed by FTIR and DSC technique. KLVFD-Cur-ME was characterized for the drug content, globule size, zeta potential, and pH. A transparent and homogeneous KLVFF-Cur-ME is achieved with a drug content of 80.25% and a globule size of 76.1 ± 2.5 nm. The pH of KLVFF-Cur-ME is 5.33 ± 0.02, indicating non-irritation to nasal tissues. KLVFD-Cur-ME does not show nasal ciliotoxicity. An ex vivo diffusion study revealed that KLVFF-Cur-ME partitions the porcine nasal mucosa through diffusion, following the Higuchi model. This investigation demonstrates the successful synthesis of a bifunctional KLVFF-Cur-ME as a novel prototype to deliver anti-A Current therapeutic agents, which are available to treat AD, fall into two categories, including drugs that only treat cognitive symptoms and drugs that may delay clinical decline by removing Aβ -carbodiimide hydrochloride (EDC), N-hydroxysulfosuccinimide (sulfo-NHS), tetrahydrofuran (THF), dimethylaminopyridine (DMAP) succinic anhydride polysorbate 80 (TWEEN 80), Sorbitan monolaurate (Span 20), glycerine, propylene glycol, polyethylene glycol 400 (PEG 400), and isopropyl myristate (IPM) were purchased from Sigma-Aldrich . Ethanol, methanol, and carbon tetrachloride were obtained from Sigma-Aldrich . Curcumin was obtained from Merck . KLVFF peptide was synthesized and purified by PEPMIC . Monobasic potassium phosphate was purchased from RCI Labscan . Sodium hydroxide was obtained from KEMAUS .Pluronic F-127® were converted to carboxyl groups according to the following procedure. Pluronic F127® was dissolved in tetrahydrofuran . Then, 4-dimethylaminopyridine , triethylamine (108 µL), and succinic anhydride (800 mg) were added. The mixture was stirred for 48 h at room temperature. The solution was dried by rotary evaporation and was dissolved in carbon tetrachloride (30 mL). The excess succinic anhydride was removed by filtration. The COOH-Pluronic F127 was purified by precipitation with ice-cold diethyl ether. The product was identified by FTIR spectroscopy. The illustration of synthetic route of carboxylated Pluronic F127 is shown in Terminal hydroxyl groups on Pluronic F127Carboxylated Pluronic was conjugated to the amino groups of KLVFF peptide by a carbodiimide reaction which is mainly used to form amide linkages between amines and carboxylates ,41. The −1. Pluronic and carboxylated Pluronic were physically mixed with potassium bromide and compressed into a disk using a Specac  hydraulic press before scanning.The FTIR spectra were obtained by using a Nicolet iS5 FTIR spectrometer , which was operated in the range of 4000–400 cmThe DSC curves of Pluronic F127, COOH-Pluronic F127, and KLVFF-Pluronic F127 were obtained using a differential scanning calorimeter  equipped with a heat flow sensor, and joined via the TA Controller TC 15 interface to a computer. Measurements were driven by Pyris™ software version 13.2.1.0007 . Samples for DSC measurements were weighed using a Mettler Toledo AT 261  microbalance (±0.01 mg) and sealed in 40 µL standard aluminum crucibles with a single hole punched in the lid. The total mass of a sample was between 3 and 5 mg. An empty pan of the same type was employed as a reference. DSC scans of each mixture were performed at a heating rate of 5 °C/min in the temperature range of 25–200 °C. The DSC cell was purged with a stream of nitrogen at a rate of 50 mL/min.g rpm for 30 min. The supernatant was diluted with ethanol. The concentration of solubilized curcumin was determined spectrophotometrically at the maximum wavelength of 465 nm. Solubility was carried out in triplicate. To find out the appropriate ratios of oils, surfactants, and co-surfactants as excipients for microemulsions (ME) formulation, the solubility of curcumin in various oils, surfactants, and co-surfactants was investigated by adding excess curcumin into 2 mL of each vehicle. The mixtures were mixed under stirring for 24 h. After equilibrium for 24 h at room temperature, samples were centrifuged at 5000× Pseudo-ternary phase diagrams were constructed to determine the region into which the maximum amount of ME formation occurred. The appropriate components from the result of the solubility study were selected to prepare ME by the spontaneous emulsification technique . SurfactFrom pseudo-ternary phase diagrams, the one showing the maximum region for ME was selected to be considered as an optimized ratio for drug-loaded ME formulation. Various formulations were investigated in terms of droplet size, zeta potential, polydispersity index (PDI), transparency, pH, and conductivity to obtain an optimized formulation. The formulation that showed satisfactory results was selected for fabricating curcumin-loaded KLVFF-Pluronic F127 microemulsions (KLVFF-Cur-ME).KLVFF-Cur-ME was prepared by dissolving curcumin into the oil phase by adding the required quantity of Smix and water and stirring to form a clear and transparent dispersion. The characterizations of KLVFF-Cur-ME were investigated for the percentage of transmittance, globule size, zeta potential, conductivity, and pH.The electrical conductivity of ME was measured with a conductivity meter  equipped with a magnetic stirrer. The conductivity measurement was performed using a conductivity cell (with a cell constant of 1.0) consisting of two platinum plates separated by desired distance and liquid between the platinum plates acting as a conductor.The average droplet size and polydispersity index (PDI) of ME were measured by dynamic light scattering . All determinations were conducted in triplicate.The percent transmittance of the system was checked by measuring transmittance at 650 nm with distilled water as a reference ,43 by a The pH values of ME and COOH-ME were determined using a digital pH meter , standardized using pH 4 and 7 buffers before use.The TEM micrograph of KLVFF-Cur-ME was obtained to characterize morphology  at an acceleration voltage of 100 kV and 8000× magnification. KLVFF-Cur-ME was stained with a 1% aqueous phosphotungstic acid solution and deposited on the carbon-coated copper grid.g rpm for 30 min. The supernatant was taken, and the amount of curcumin was analyzed by a UV–visible spectrophotometer [The curcumin content in KLVFF-Cur-ME was determined using a spectrophotometer at the maximum wavelength of 465 nm. A certain amount of KLVFF-Cur-ME was diluted with ethanol, followed by centrifugation at 5000× otometer .Curcumin permeation study was conducted using Franz diffusion cells  through the porcine nasal mucosa. Freshly excised porcine nasal mucosa, obtained from the slaughterhouse, was immediately soaked in phosphate buffer (pH 6.4) The protocol for the use of cadavers was approved by the Animal Care and Use Committee, Faculty of Veterinary Medicine, Chiang Mai University, Thailand (FVM-CMU-ICUC Ref. No. R5/2563). Fresh porcine nasal mucosa with a thickness of 0.2 mm was mounted between donor and receptor compartments with a volume capacity of 12.5 mL. KLVFF-Cur-ME equivalent to a similar amount of curcumin in the solution was placed into the donor compartment. At the same time, the receptor was filled with phosphate-buffered saline , which was maintained at 37 °C under continuous stirring. A one milliliter aliquot was taken at different time intervals and replaced with an equal volume of PBS. After suitable dilution, the sample was analyzed for curcumin content by HPLC.v:v) mixture of acetonitrile and 2% acetic acid in water. The UV detector was set at a wavelength of 424 nm.The samples obtained from the receptor compartment of Franz diffusion cell were analyzed by HPLC. The separation was performed on a reverse-phase C18 column . The elution was carried out with isocratic solvent systems with a flow rate of 1.2 mL/min at ambient temperature. The mobile phase consisted of a 50:50  with a magnification of 10×, captured to evaluate any damage to the nasal mucosa. The protocol for the use of cadavers was approved by the Animal Care and Use Committee, Faculty of Veterinary Medicine, Chiang Mai University, Thailand (FVM-CMU-ICUC Ref. No. R5/2563).Freshly excised porcine nasal mucosa was obtained from the slaughterhouse and immediately soaked in phosphate buffer (pH 6.4). The cartilage was gently removed to isolate nasal mucosa. Each piece of the porcine nasal mucosa with even thickness (0.2 mm) was mounted on Franz diffusion cell with the positive control  negative control (PBS pH 6.4) and KLVFF-Cur-ME for 2 h. After that, all pieces of mucosa were rinsed with PBS (pH 6.4) and soaked in a 10% n = 3 experiments. t-test was used to determine a significant difference between the means of the two groups. Statistical analysis of data was completed using an analysis of variance (one-way ANOVA), followed by Newman–Keuls method as a post hoc test to evaluate the significance of differences. In all cases, a value of p < 0.05 was considered statistically significant. All data were presented as mean ± SEM, −1 [−1, centered at around 2882 cm−1 [−1 [−1 and 1556 cm−1, respectively [−1 [−1 arising from the C=O stretching vibration [Carboxylated Pluronic F127 (COOH-Pluronic F127) was derivatized through the reaction of Pluronic-OH with succinic anhydride described in −1 . In cont−1 . Moreove882 cm−1 , and thecm−1 [−1  for COOHectively . The strvely [−1  and an oibration  were obsDifferential scanning calorimetry could be used to determine the phase state of a compound. It can also be used to observe the fusion and crystallization characteristics of polymers . After iThe solubility of curcumin in various oils and co-surfactants was analyzed to screen the components for microemulsions. Selecting the best oil phase was required for the maximum solubility potential for the drug to cover a larger ME region in the ternary plots and obtain a stable ME ,52. SoluThe pseudo-ternary phase was constructed from the components that showed the maximum solubility, as shown in Physicochemical and formulation factors are necessary for the rational design of a dosage form . The expNormally, a PDI of close to zero indicates the uniformity of a globule size, while one indicates a polydisperse sample with multiple size populations . Values The morphology of KLVFF-Cur-ME using TEM revealed a spherical shape with size in the nanoscale (20–65 nm), as shown in 2r) for the zero-order, first-order, and Higuchi models are shown in 2r = 0.9222), indicating a curcumin partitioning through diffusion, since the porcine mucosa acted as a barrier or controlling membrane [An ex vivo diffusion study was performed to assess the drug diffusion through a biological membrane simulating the actual in vivo barrier to drug diffusion . The ex membrane . The resmembrane . MoreoveThe nasal ciliotoxicity was studied to evaluate the toxic effect of the excipients used for the nasal mucosa formulations. β aggregation via intranasal administration. However, the potential of the developed KLVFF-Cur-ME for nose-to-brain delivery of curcumin can only be established after in vivo toxicity and biodistribution studies.In this study, Cur-ME was prepared using the titration method. A carbodiimide reaction was performed for the KLVFF conjugation. KLVFF-Cur-ME was successfully synthesized with acceptable physicochemical characteristics and was suitable for intranasal administration. The encapsulation of curcumin into the microemulsions improved the solubility and released the profile of curcumin. KLVFF-Cur-ME did not show nasal ciliotoxicity. The kinetic release profile of KLVFF-Cur-ME followed the Higuchi model. The result from the ex vivo permeation study suggested the successful diffusion of KLVFF-Cur-ME through the porcine nasal mucosa. Therefore, KLVFF-Cur-ME may provide a new approach for inhibiting A


Long non-coding RNAs (lncRNAs) display regulatory function flexibly in tumor onset and developments. Our study aimed to delve into the roles of lncRNA LINC01569 (LINC01569) in colorectal cancer (CRC) progression to study the potential mechanisms.The genetic expression profiles of miR-381-3p and LINC01569 were measured by RT-PCR. The subcellular localization of LINC01569 in CRC cells was identified using subcellular fractionation location. Loss-of-function assays were performed to explore the potential effects of LINC01569 on CRC progression. Dual-luciferase reporter analysis was employed to verify the binding connections among LINC01569, miR-381-3p, and RAP2A.LINC01569 expression was distinctly increased in CRC. Curiously, if LINC01569 is removed, CRC cells will not migrate, proliferate, and invade remarkably. Molecular mechanism exploration uncovered that LINC01569 acted as a ceRNA competing with RAP2A to bind with miR-381-3p. Furthermore, rescue experiments corroborated the fact that miR-381-3p suppression reversed the inhibitory actions of LINC01569 knockdown on the expression of RAP2A and CRC progression.Overall, our findings indicate that LINC01569 plays a key role in CRC development by means of aiming at the miR-381-3p/RAP2A axis and can be equivalent to an underlying medicinal target to save CRC patients. Colorectal cancer (CRC) is one of the most common types of malignancy and the third leading cause of cancer-associated mortality worldwide . More thLong non-coding RNAs (lncRNAs) belong to a type of RNAs without coding capacity . In mammLncRNA LINC01569 (LINC01569), located at 16p13.3, is a novel lncRNA whose function in vital movement remains largely unclear. Previously, LINC01569 was shown to mediate glucocorticoid effects on mechanotransduction by destabilizing messenger RNA (mRNA) of mechanosensors  along with the colonic epithelial cell line NCM460 from National Biomedical Laboratory . In medium RPMI-1640  supplemented with 10% FBS , 100 U/ml penicillin and 100 mg/ml streptomycin), cells were cultivated. Cells were kept within the incubator at 5% COGenomics substances were provided by Liaoning Biology , including negative control (NC) plasmids, miR-381-3p inhibitors, miR-381-3p mimics, and short hairpin RNA for LINC01569 (sh-LINC01569-1), and LINC01569 (sh-LINC01569-2). The transfections of all substances into SW620 and HCT116 cells were carried out for 48 h using Lipofectamine 2000 Reagent .-ΔΔCt method. Primer sequences were shown in Following the standard process, TRIzol reagent  was applied for the extraction of total RNAs from clinical samples and human specimens. Following the instructions of the manufacturer, total RNAs were transcribed reversely into cDNA by utilizing a PrimeScript™ RT reagent kit . RT-PCR was performed by applying ABI 7900HT Fast Real-Time PCR System. GAPDH or U6 served as the endogenous control, and relative expression was compared by the 2For CCK-8 assays, cells were plated into plates with 96 wells and cultivated according to the indicated days. Subsequently, CCK-8 solution was added and incubated for 2 h. An absorbance of 450 nm was eventually recorded by utilizing a microplate reader. For colony formation assays, cells were plated into plates with 6 wells and cultured for 14 days. Subsequently, methanol was used to fix clones, followed by dyeing with 0.1% crystal violet. Finally, our group calculated the colony numbers.2 with a temperature of 37°C for 48 h, the 1 × 104 cells were seeded into plates with 96 wells. Every well with cells cultivated for as long as 2 h was added with Edu solution with a 50 μM of eventual concentration. After that, cells were fixed by using 4% paraformaldehyde and dyed by applying Apollo dye solution for proliferation exploration. By using a fluorescence microscope, the observation, calculation, and photographing of the cells with positive EdU were carried out after we washed the cells with PBS .By using EdU (5-ethynyl-20-deoxyuridine) analysis resulting from Cell-Light EdU DNA Cell Proliferation Kit, we surveyed the proliferation of cells. Cultured at 5% CO5 density) were seeded into plates with 12 wells and grown until reaching 80% confluence. Subsequently, the linear scratch of the cell monolayer was carried out by using the tip of a sterile pipette. At 0 and 48 h post-wounding, 5% paraformaldehyde was used to fix the cells for a quarter followed by image recordings for the calculation of the migratory cells.The SW620 and HCT116 cells  was used. After the upper chamber was coated with Matrigel, cells were plated onto them. The chamber at the bottom was provided with the complete medium which carried 10% FBS . After 24 h, the lower chamber was provided with crystal violet at a concentration of 0.1 and 20% methanol. Under the microscope, we calculated the cells of invasion.To separate the RNAs from nuclear or cytoplasm fraction, our group used a Nuclear/Cytosol Fractionation Kit . The levels of LINC01569 in cytoplasm or nuclear components were examined using subcellular fractionation location assays . GAPDH a2/2), volume was calculated. All mice were grown for 28 days, followed by sacrifice for the collection of tumor specimens.Abiding by the accord accepted by the Animal Care and Use Committee of Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, animal experiments were conducted to explore the function of LINC01569 knockdown. Briefly, 6 × 105 cells with sh-LINC01569 or sh-NC stable transfection were injected into the flank of nude mice (n = 6 per group). Every 7 days, our group measured the width and length of tumors. According to the formula (length × (width)via the use of PCR, the mutant types (MUT) along with the wild ones (WT) of RAP2A, which carried the assumed binding area to couple with miR-381-3p, were duplicated into the reporter vector of pmirGLO luciferase . Then, cells were co-transfected with miR-381-3p mimics or miR-NC and luciferase vector containing 3′-UTR of RAP2A, wild-type (Wt) or mutant (Mut) RAP2A fragment, using Lipofectamine 2000. Luciferase reporter assay was performed using the Dual-Luciferase Reporter Assay System (Promega) 48 h later.Enlarged via SDS-PAGE at a density of 10%, then cultivated overnight along with primary antibodies at a temperature of 4°C to go against PAP2A. The primary antibodies and second antibodies were purchased from CWBIO . Finally, ECL chemiluminescence kit (Beyotime) was used to visualize blots.Through the application of RIPA lysis buffer which carried proteinase restrainer, we acquired and lysed SW620 and HCT116 cell lines . By applying the BCA protein analysis tool, we quantitated the overall protein . Before incubating with secondary antibodies linked to HRP, proteins blotted onto PVDF membrane were severed All statistical analyses were performed using SPSS 18.0 software (IBM) and Prism 7 . All assays were conducted in triplicate. Differences between two independent groups were tested with the Student’s t-test. The one-way ANOVA was used to analyze three or more groups. A p-value <0.05 was considered statistically significant.Primarily, whether LINC01569 levels were dysregulated in CRC was examined by using RT-PCR. via loss-of-function methods. The interference potency of two shRNAs was displayed in in vivo assays. The volume and weight of tumors in the LINC01569 (LINC01569 knockdown) group were significantly smaller than that in the control group  of LINC01569 were screened from Starbase 2.0 and miRCode database  To find the mRNAs that were involved in LINC01569/miR-381-3p regulation of CRC activity, Starbase 2.0 was used to find the possible genes. We paid attention to RAP2A which had been demonstrated to play a role of a tumor promoter for several tumors, including CRC , 18. A dTo further explore the biological interactions among LINC01569, miR-381-3p, and RAP2A in CRC, SW620 and HCT116 cells were co-transfected with sh-LINC01569 and miR-381-3p inhibitor. As presented in in vivo and in vitro assays , a protein regarding Ras, presented as one of the massive family of small GTPase protein and a p53 transcription target , 32. Morby RAP2A . In CRC,by RAP2A . In addiWe acknowledged some limitations in this study. First, the sample size was relatively small; we will collect more samples to explore the clinical significance of LINC01569 in CRC patients in future studies. Second, we did not explore the potential mechanisms involved in LINC01569 overexpression in CRC.Our study identified a novel lncRNA LINC01569 associated with poor prognosis in CRC. Upregulation of LINC01569 facilitated the capability to proliferate and metastasize for CRC cells by targeting miR-381-3p/RAP2A. Therefore, the targeted regulation of the LINC01569/miR-381-3p/RAP2A axis improved the condition of the disease.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University. The patients/participants provided their written informed consent to participate in this study. The animal study was reviewed and approved by Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University.G-yY and Z-zZ wrote the main manuscript and analyzed the data. C-cZ, G-yY, Z-jC, ZC, and LC performed the experiments. G-yY, Z-zZ, and GZ designed the study. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Cydia pomonella: two Cydia pomonella granulovirus genotypes, CpGV-M and CpGV-R5, and Cryptophlebia peltastica nucleopolyhedrovirus (CrpeNPV). The influence of the order of ingestion of the virus genotypes, the existence of an ingestion delay between the genotypes and the specificity of each genotype involved in the success of multiple infection were studied in the case of Cydia pomonella resistance. To obtain a multiple infection in resistant insects, the order of ingestion is a key factor, but the delay for ingestion of the second virus is not. CrpeNPV cannot substitute CpGV-R5 to allow replication of CpGV-M.The genetic diversity of baculoviruses provides a sustainable agronomic solution when resistance to biopesticides seems to be on the rise. This genetic diversity promotes insect infection by several genotypes  that are more likely to kill the host. However, the mechanism and regulation of these virus interactions are still poorly understood. In this article, we focused on baculoviruses infecting the codling moth,  The use of pathogens is a well-known approach for biological control of insect pests. Among them, members of the baculovirus family are widely used as crop protection products ,2.Alphabaculovirus, Gammabaculovirus and Deltabaculovirus, previously classified as nucleopolyhedroviruses (NPVs), while granules are found only in the Betabaculovirus that are also called granuloviruses (GVs). Polyhedra contain tens of virions while granules usually contain one (or sometimes more) virion [The most common way of infection for baculovirus-based biocontrol products is the oral ingestion of the virus by host larvae. Baculoviruses produce occlusion bodies (OBs) that facilitate the survival and transmission of the virus from one host to another. Two types of OB can be found in baculovirus, polyhedra and granules. Polyhedra are found in the genera ) virion ,4. When ) virion . Each nuBaculoviruses show a high level of genetic diversity. In a given virus population, diverse genotypes can be found. In addition, geographically distant virus populations can differ not only in the genotypes present, but also in their respective frequencies ,7.Spodoptera frugiperda MNPV (SfMNPV), or Autographa californica MNPV (AcMNPV), it has been demonstrated that various genotypes can be occluded in a single OB [In various MNPV, like ingle OB . This obingle OB . In suchingle OB .The various genotypes of a virus population differ in their biological characteristics, particularly the ability to overcome host defense mechanisms ,11. GenePhthorimaea operculella (Zeller) (Lepidoptera: Gelechiidae) it has been shown that some granulovirus isolates, which are mixtures of genotypes, have a higher pathogenicity than isolates composed of pure genotypes. It has also been shown that such association of different Phthorimaea operculella granulovirus (PhopGV) genotypes does not result in a “hybrid” genotype but that genotypes are maintained in stable frequencies over time [In SfMNPV, the genetic diversity of isolates is maintained and finely regulated during the different generations to guarantee the best efficiency. In multiple SfMNPV infections, each genotype appears to contribute to preserve the survival of the whole population ,17,18. Iver time ,20.Betabaculovirus) used as a bioinsecticide worldwide in the biological control of the codling moth, Cydia pomonella (Linnaeus) (Lepidoptera: Tortricidae), a pest of apples and pears. The first isolate of CpGV was found in Mexico. It is composed of a single genotype called CpGV-M. This isolate was used to develop commercial bioinsecticides extensively used in Europe since their approval in the nineties. However, after years of using CpGV-M, codling moth populations have developed resistance [CpGV is a granulovirus  were allowed to feed on mixtures of CpGV-M and CpGV-R5, successful replication of CpGV-M was detected. Surprisingly, these mixed virus populations appear to be more effective in controlling RGV resistant individuals than CpGV-R5 alone [Five types of resistance in escribed . They diescribed . In laboR5 alone . SimilarR5 alone . In our Cryptophlebia peltastica nucleopolyhedrovirus (CrpeNPV) [Once the order and the delay of ingestion was characterized, it was necessary to determine (i) if the help needed for CpGV-M replication in a non-permissive insect is generic, for example by weakening the host, making it permissive to a second infection, regardless of the first pathogen, or (ii) the help provided to CpGV-M is specific to CpGV-R5. To that end, we tested the ability of another baculovirus to act as helper for CpGV-M. The CrpeNPV)  is able CrpeNPV) , therefo®. CpNPP is a colony susceptible to CpGV-M and CpGV-R5 [CpNPP is the reference insect colony susceptible to CpGV-M and was provided by Natural Plant Protection . It originates from Northern France, and is used for the industrial production of Carpovirusine CpGV-R5 . The susGV insect colony is resistant to CpGV-M. This colony originates from a natural insect population collected in the field at Saint-Andiol (St-A) in the Bouches-du-Rhône region of France. Its resistance is characterized as dominant and carried by the Z sex chromosome (type I resistance). This colony remains susceptible to other isolates, such as CpGV-R5 [The R CpGV-R5 .50s are 13.10 (6.55–23.20), and 6.76 (2.6–13.37) OB·µL, respectively, while in RGV, CpGV-M and CpGV-R5 LD50s are 2.22 × 106 (1.19 × 106–5.67 × 106), and 22.43 (13.73–34.36) OB·µL, respectively [CpGV-M is the first recorded CpGV isolate, discovered in Mexico and described by Tanada in 1964 . Its genectively .C. peltastica (litchi moth). It has a genome of 115,728 bp (GenBank accession numbers: MH394321) [CrpeNPV is an SNPV isolated in 2018 in South Africa from H394321) . The CrpOB concentrations were estimated by counting on dark field light microscopy as previously described .GV larvae. Three independent assays were carried out. CpGV-M and CpGV-R5 viruses were deposited on different dishes of Stonefly Heliothis medium (20 cm²) at a concentration of 104 OB·cm−2. Neonate larvae were placed onto the surface of the 1st and then the 2nd virus treated medium (CpGV-M or CpGV-R5), for various durations . The larvae were then transferred to non-treated medium for 3 days. The larvae were then crushed individually and the virus resuspended into 200 µL of distilled water and analyzed by quantitative polymerase chain reaction (qPCR) coupled to high-resolution melting (qPCR/HRM)  see  below.GV neonate larvae were first allowed to feed on CpGV-R5 treated medium for 30 min (time necessary for ingestion of sufficient virus to result in double infection [Rnfection ). LarvaeGV was first verified (data not shown).It has been demonstrated that CrpeNPV is able to infect various codling moth colonies . The abi4 OB·cm−2. At 3 days, the larvae were collected and crushed into 200 µL. Each sample was split into two separate qPCR reactions and each amplicon was identified by HRM analysis.To check if double infections can occur using two virus species, neonate CpNPP larvae were exposed to medium (Stonefly Heliothis medium) inoculated with a virus mixture , with final concentration of 10GV neonate larvae were fed with a mixture of 50% CrpeNPV and 50% CpGV-M, and processed under similar conditions. Similarly, RGV third instars were fed with a mixture of 50% CrpeNPV and 50% CpGV-M with 2.22 × 104 OB·cm−2, collected at 4 days and processed under similar conditions.Lastly, to check if CrpeNPV can substitute CpGV-R5 for helping CpGV-M replication in resistant larvae, Rpe38 gene region using the primers CpGV-18734F (5′-GCCACCATTAGTGAATCATC-3′) and the reverse primer CpGV-18855R (5′-TAAGTCAGGACACCCAAACC-3′). The pe38 gene is involved in type I resistance of C. pomonella [qPCR was carried out in the conditions previously described . CpGV-M CrpeNPV was identified using the primers polh_CrpeNPV_F (5′-CGAGCATGAAATCGAGGAAC-3′) and polh_CrpeNPV_R (5′-ACTTCGTGAGGCACATAGTC-3′) targeting the polyhedrin region (GenBank accession no.MH394321) . CrpeNPVEach amplicon produced by the different viruses was submitted to HRM analysis. A melting curve step was added: 5 s at 95 °C then 10 s at 50 °C and increasing the temperature from 70 to 90 °C by steps of 0.2 °C, maintained for 10 s each. The software Biorad CFX maestro™ and Precision Melt Analysis™ were used for interpretation. To avoid possible interferences, amplifications of CpGV and CrpeNPV were carried out separately.GV larvae are resistant to the CpGV-M isolate and none were found infected with CpGV-M alone; they are susceptible to the CpGV-R5 isolate. In our experimental conditions, the proportion of larvae infected with CpGV-R5 varied from 50% to 80%, regardless of the order of ingestion.CpNPP larvae are susceptible to all viruses tested. RGV, when CpGV-M was ingested first, followed by CpGV-R5, no CpGV-M infection was observed. Conversely, if CpGV-R5 was ingested first, 5% to 20% of individuals were infected with both viruses  larvae were allowed to feed on mixtures of CrpeNPV and CpGV-M. The replication of each virus was detected using qPCR followed by HRM. The mean Tm (melting temperature) of CpGV-M and CrpeNPV amplicons were 81.8 °C and 84.4 °C, respectively, and their size was verified by electrophoresis (data not shown). The two peaks are clearly distinct, allowing non ambiguous identification of each infection. Each larva was analyzed separately. Replication of GV and NPV was detected in more than 70% of the CpNPP infected larvae, for both CpGV genotypes . As shown for CpNPP, it was found that RGV larvae are permissive to multiple infections.When RGV larvae were inoculated with CpGV-M and CrpeNPV-SA isolates. No RGV larva was found infected with CpGV-M in the presence of CrpeNPV, even when doubling the quantity of CpGV-M. Given the sensitivity of qPCR approaches, if CpGV-M replication had occurred, it certainly would have been detected. To check if the development stage was a factor influencing the multiple infection, a similar experiment was carried out with third instar RGV larvae. No double infection was detected.Finally, neonate RPseudaletia unipuncta, while the NPV alone could not replicate [trans complementation manner [Spodoptera littoralis NPV (SpliNPV). pif-1 deficient genomes can infect orally only if the virions contain active PIF-1 protein, that is, a cis complementation. Co-infection allowed pif-1 deficient genomes to be enveloped with PIF-1 containing membranes due to the presence in the same cell of complete genomes [Helper activities have been previously described in other baculovirus/host systems. Tanada (1959) described how the presence of a GV allowed replication of PsunNPV in the armyworm eplicate . Later in manner ,41,42. Sn manner . A diffe genomes .The point of blockage of CpGV-M replication on type I resistant codling moth larvae has been studied by Asser-Kaiser et al. (2011). Replication is blocked independently of the mode of entry, by ingestion or by injection. CpGV-M is able to enter into the cells, but its replication is stopped at an early stage prior to DNA replication. These authors concluded that the resistance is not restricted to the primary infection in the midgut, but all cells in the larvae appear to be restrictive for the replication of CpGV-M .GV host [Previous work on multiple infections has shown that the CpGV-R5 isolate allows replication of the CpGV-M isolate in the non-permissive RGV host . Our preGV host . It can 4 OB·µL) that do not reach 100% mortality to avoid such a massive midgut infection.If a midgut cell needs to be infected by CpGV-R5 prior to CpGV-M co-infection to allow replication of the latter, the total number of midgut cells infected should be very important to result in 10% to 20% double infections , even ifIn the systems that have been studied, midgut infection appears to be transient, that is, infected cells are lost by sloughing and no infection from cell to cell was observed in the midgut ,46,47. TCydia pomonella/CpGV model and with the tools currently available, it is only possible to determine the duration of the ingestion delay allowing a double infection and not the existence of a superinfection time-lapse, which is only accessible at the cell scale. For these hypotheses to be tested, it will be necessary to follow the infection of each individual virion in the midgut. Such an observation will be possible once the ANCHORTM system we have previously adapted to AcMNPV [In cell culture, it has already been shown for SfMNPV that the probability of a successful infection with a second virus is related to the time-lapse between the first and second infection, referred to as the window of superinfection . The timo AcMNPV  is transC. pomonella larvae, carrying diverse resistance genes to CpGV, CpS, CpRR1, CpR5M, and CpRGO colonies [GV, this virus also replicates efficiently. We have used this second virus species (CrpeNPV) to test if the helper effect of CpGV-R5 could be substituted by another virus that replicates in the larvae. We first determined that double infections can occur using CrpeNPV and CpGV-M in fully permissive CpNPP larvae and CrpeNPV and CpGV-R5 in RGV larvae. Subsequently, the occurrence of double infection was analyzed when a mixture of CrpeNPV and CpGV-M was given to RGV larvae. No double infections were found, suggesting that CrpeNPV cannot help CpGV-M to replicate. Probably other CpGV genotypes could act as helpers, but this has not yet been tested.It has been demonstrated that CrpeNPV is able to replicate in four different groups of colonies . We haveGV cells susceptible to CpGV-M. This theory would explain the results observed in the present experiments. If such a factor exists, the absence of helper activity of CrpeNPV would suggest that it is specific.We have previously proposed  that a dCodling moth resistance to CpGV genotypes is a major threat for apple and pear producers. The presence of multiple CpGV genotypes in the virus inoculum results in it not behaving as single genotypes do. The interactions between CpGV genotypes appear to be specific and cannot be substituted by the presence of more distant viruses infecting the same host. The analysis of these interactions opens the way to a more detailed understanding of the early steps of the infection process.